Part:BBa_K4593004
Characterization device for P2 promoter in E.coli
Usage and Biology
The QS System is a mechanism employed by specific bacteria to sense their population density and subsequently regulate gene expression accordingly. In the case of S. aureus, it produces a signaling molecule known as autoinducing peptide (AIP) during its growth. AIPs are recognized by a membrane receptor called AgrC. AgrC, in turn, phosphorylates AgrA, leading to the activation of the downstream promoter P2. (Marchand & Collins, 2013)
This part is constructed for the characterization of Promoter P2 in E. coli, in which sfGFP downstream of the P2 promoter will be expressed under the presence of AIPs. However, it should be noted that this part does NOT work as expected!
Team:BNDS-China 2023
Design of the plasmid
Initially, our objective is to confirm the inducibility of the P2 promoter by AIPs. In our plasmid design, sfGFP serves as a reporter to indicate whether AgrA activated the transcription of genes downstream P2 promoter in the presence of AIPs. Consequently, the presence of observable fluorescence would signify the effective activation of the P2 promoter, validating its utility in our project. The plasmid design is depicted below.
Figure 1. Design of P2 characterization plasmid.
Construction of characterization plasmid
The PCR is employed to amplify our plasmid; the AgrC and AgrA were directly amplified from the plasmid pXylA-agrCA-I obtained from Addgene. [1]. P2 promoter was synthesized according to the previous existing sequence (Part: BBa_K1960900) that iGEM 16_XMU-China designed. The sfGFP was obtained from 2002p derived from BNDS-China 2021. All the fragments were added to the pET28a (+) backbone through Goldengate Assembly. The plasmid is transformed into TOP 10 competent cells, and the cells are spread on the LB agar plate with K+. After individual clones are selected and allowed to shake overnight, the plasmid is extracted and sent for sequencing, with the outcome being confirmed as correct.
Figure 2. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length. Lane 1: 2428bp; Lane 2: 4373bp.
The RBS is directly synthesized according to the previous existing sequence (Part: BBa_B0034), and PCR is employed to amplify the plasmid parts(P1 and P2) to add RBS downstream of the promoter. The plasmid underwent the same procedure, transformed into TOP 10 competent cells, and was sent for sequencing, with the outcome being confirmed as correct.
Figure 3. The AGE result of the PCR product for constructing the characterization plasmid. The bands indicate the correctness of each component’s length.
The extracted plasmid is introduced into BL21 to enable the expression of sfGFP. After an overnight incubation period, IPTG is introduced to induce expression. In conditions where S. aureus is not present, we observed the expression of sfGFP. This observation suggests that P2 is not induced but rather expressed constitutively. As a result, it becomes evident that P2 is not functioning as originally intended in our experiment, and a redesign of the gene circuit is required.
Figure.3 The picture of the induced BL21. Fluorescence can be observed in the absence of AIPs.
To determine the cause of the failure, the dry lab component was employed, and the transcriptional rates of the P2 promoter in E. coli were assessed using the De novo DNA calculator (LaFleur et al., 2022). The result is shown below.
Figure 4. The predicted transcription rates of p2 specifically in E. coli.
The calculated result reveals that it is evident that the transcription rate at the beginning of the sequence is exceptionally high even without an activator, which is consistent with the wet lab result.
Two main reasons contribute to the failure of our design. First, the structure of RNA polymerase and the microenvironment for transcription is very different in E. coli and S. aureus, causing the transcription activity of P2 to be exceptionally high. Also, as E. coli is a gram-negative bacteria and S. aureus is a gram-positive bacteria, the membrane protein AgrC may not locate itself successfully on the cell membrane of E. coli, causing the signal transduction to fail. For those reasons, we decided to move the QS detection module to a gram-positive bacteria, Bacillus subtilis, which has more shared features with S. aureus (see part BBa_ ).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 883
Illegal NheI site found at 906 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1294
Illegal BamHI site found at 2178 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 121
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